Mutagenesis of plants overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes

Abstract

CONSTANS (CO) promotes flowering of Arabidopsis in response to long photoperiods. Transgenic plants carrying CO fused with the cauliflower mosaic virus 35S promoter (35S::CO) flowered earlier than did the wild type and were almost completely insensitive to length of day. Genes required for CO to promote flowering were identified by screening for mutations that suppress the effect of 35S::CO. Four mutations were identified that partially suppressed the early-flowering phenotype caused by 35S::CO. One of these mutations, suppressor of overexpression of CO 1 (soc1), defines a new locus, demonstrating that the mutagenesis approach is effective in identifying novel flowering-time mutations. The other three suppressor mutations are allelic with previously described mutations that cause late flowering. Two of them are alleles of ft, indicating that FT is required for CO to promote early flowering and most likely acts after CO in the hierarchy of flowering-time genes. The fourth suppressor mutation is an allele of fwa, and fwa soc1 35S::CO plants flowered at approximately the same time as co mutants, suggesting that a combination of fwa and soc1 abolishes the promotion of flowering by CO. Besides delaying flowering, fwa acted synergistically with 35S::CO to repress floral development after bolting. The latter phenotype was not shown by any of the progenitors and was most probably caused by a reduction in the function of LEAFY. These genetic interactions suggest models for how CO, FWA, FT, and SOC1 interact during the transition to flowering.

Figure 1.

Detection of CO mRNA in Wild-Type and Transgenic Plants. (A) Total RNA extracted from wild-type Ler (WT) and transgenic plants carrying 35S::CO:GR (CO:GR) or five transformants carrying 35S::CO (lanes 1 to 5) was transferred to a filter and hybridized with the CO cDNA. (B) As a control for loading, the filter was stripped and hybridized with a fragment derived from the tubulin gene (see Methods).

Figure 2.

Phenotype of 35S::CO and 35S::CO Plants Carrying Suppressor Mutations. (A) A 3-week-old wild-type (WT) plant grown under long days (LDs) (left), a 35S::CO plant grown under long days (center), and a 35S::CO plant grown under short days (SDs) (right). (B) The inflorescence apex and the pistil-like structure formed at the apex of the shoot of a 35S::CO plant (arrow). (C) Siliques of wild-type (left) and 35S::CO (center and right) plants. (D) A 3-week-old 35S::CO plant (left), a soc1 35S::CO plant (center), and a ft-7 35S::CO plant (originally designated suppressor mutant 1) (right) grown under long-day conditions.

Figure 3.

Segregation of the Late-Flowering Phenotype in the F₂ Population of a Cross between soc1 35S::CO and fwa-1/+ 35S::CO Plants. Plants were grown under long-day conditions. Plants showing severe (fwa-1/fwa-1 35S::CO–like) and weak (fwa-1/+ 35S::CO–like) floral meristem identity defect segregated in the population and are indicated by the black and cross-hatched bars, respectively. Plants showing a normal 35S::CO–like shoot are indicated by the open bars. The open circles and horizontal lines indicate the average and the range, respectively, of rosette leaf numbers of defined genotypes grown under the same conditions. The plants flowering with between 14 and 18 rosette leaves are presumed to have the genotype soc1/soc1 fwa-1/fwa-1 35S::CO and represent approximately one in 17 of the total population.

Figure 4.

Shoot and Flower Phenotype of fwa 35S::CO Plants. All plants were grown under long-day conditions except for the plant shown in (F). (A) A 35S::CO plant (left), an fwa-100/+ 35S::CO plant (center), and an fwa-100/fwa-100 35S::CO plant (right). All plants are 1 month old. (B) A 5-week-old fwa-1/fwa-1 35S::CO plant. (C) A 2-month-old fwa-1/fwa-1 35S::CO plant showing no obvious floral organs, except occasional carpelloid structures at the apex of lateral shoots. (D) Top view of the apex of the primary inflorescence of a 1-month-old fwa-1/fwa-1 35S::CO plant. (E) Structure of the apex of a lateral branch of a 2-month-old fwa-100/fwa-100 35S::CO plant. A few carpel-like structures are visible among the leaves. (F) Structure of the apex of a lateral branch of a 2-month-old fwa-100/fwa-100 35S::CO plant grown under short-day conditions. (G) A fwa-100/+ 35S::CO flower with leaflike sepals and a secondary flower. (H) A fwa-100/fwa-100 flower with sepal/petal chimeric organs (arrows).

Figure 5.

Effects of 35S::LFY and 35S::AP1 on the fwa 35S::CO Phenotype and the Effects of Dexamethasone Applications on 35S::CO:GR fwa Plants. All plants were grown under long-day conditions. (A) An 18-day-old 35S::LFY plant showing a terminal flower phenotype. (B) An 18-day-old fwa-1/+ 35S::CO/+ 35S::LFY/+ plant showing a terminal flower phenotype. (C) An 18-day-old 35S::AP1 plant showing a terminal flower phenotype. (D) A 5-week-old fwa-1/+ 35S::CO/+ 35S::AP1/+ plant. (E) Top view of a 6-week-old fwa-1/+ co-2/+ plant treated with dexamethasone 3 weeks after sowing. (F) Top view of a 6-week-old fwa-1/+ 35S::CO:GR co-2/+ plant not treated with dexamethasone. (G) Top view of a 6-week-old fwa-1/+ 35S::CO:GR co-2/+ plant treated with dexamethasone 3 weeks after sowing.

Figure 6.

Schematic Model for the Interaction of Genes in the Control of Flowering Time and Floral Meristem Identity. (A) Model for control of flowering time. (B) Model for control of floral meristem identity (FMI). The thin numbered lines represent two possibilities, described in the text, for how the interaction between 35S::CO and fwa might inhibit LFY function. In both (A) and (B), arrows represent promotive interactions and T-bars represent inhibitory interactions. The proposed inhibitory function of FWA is based on the assumption that fwa mutations are gain-of-function mutations.